Pharmacophore identification of c-Myc inhibitor 10074-G5

Article abstract of DOI:10.1016/j.bmcl.2012.10.013

A structure-activity relationship (SAR) study of the c-Myc (Myc) inhibitor 10074-G5 (N-([1,1′-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4- amine, 1) - which targets a hydrophobic domain of the Myc oncoprotein that is flanked by arginine residues - was executed in order to determine its pharmacophore. Whilst the 7-nitrobenzofurazan was found to be critical for inhibitory activity, the ortho-biphenyl could be replaced with a para-carboxyphenyl group to furnish the new inhibitor JY-3-094 (3q). Around five times as potent as the lead with an IC₅₀ of 33 μM for disruption of the Myc-Max heterodimer, JY-3-094 demonstrated excellent selectivity over Max-Max homodimers, with no apparent effect at 100 μM. Importantly, the carboxylic acid of JY-3-094 improves the physicochemical properties of the lead compound, which will facilitate the incorporation of additional hydrophobicity that might enhance Myc inhibitory activity further still.

Full text of DOI:10.1016/j.bmcl.2012.10.013

Bioorganic & Medicinal Chemistry Letters 23 (2013) 370–374
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pharmacophore identification of c-Myc inhibitor 10074-G5
Jeremy L. Yap a, , Huabo Wang b, , Angela Hu b, Jay Chauhan a, Kwan-Young Jung a, Robert B. Gharavi c,
Edward V. Prochownik b,d, Steven Fletcher a,e,
⇑
a Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 N Pine St, Baltimore, MD 21201, USA
b Division of Hematology/Oncology, Children’s Hospital of Pittsburgh, 4401 Penn Ave, Pittsburgh, PA 15224, USA
c PharmD Program, University of Maryland School of Pharmacy, 20 N Pine St, Baltimore, MD 21201, USA
d Department of Microbiology and Molecular Genetics, The University of Pittsburgh Medical Center and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15201, USA
e University of Maryland Marlene and Stewart Greenebaum Cancer Center, 22 S Greene St, Baltimore, MD 21201, USA
a r t i c l e i n f o
a b s t r a c t
A structure–activity relationship (SAR) study of the c-Myc (Myc) inhibitor 10074-G5 (N-([1,10-biphenyl]-2-
yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine, 1) – which targets a hydrophobic domain of the Myc onco-
protein that is flanked by arginine residues – was executed in order to determine its pharmacophore. Whilst
the 7-nitrobenzofurazan was found to be critical for inhibitory activity, the ortho-biphenyl could be
replaced with a para-carboxyphenyl group to furnish the new inhibitor JY-3-094 (3q). Around five times
Article history:
Received 24 August 2012
Revised 26 September 2012
Accepted 1 October 2012
Available online 17 October 2012
as potent as the lead with an IC50 of 33
demonstrated excellent selectivity over Max–Max homodimers, with no apparent effect at 100
Importantly, the carboxylic acid of JY-3-094 improves the physicochemical properties of the lead
compound, which will facilitate the incorporation of additional hydrophobicity that might enhance Myc
inhibitory activity further still.
l
M for disruption of the Myc–Max heterodimer, JY-3-094
M.
Keywords:
Myc
Max
10074-G5
Pharmacophore
Cancer
l
Ó 2012 Elsevier Ltd. All rights reserved.
c-Myc (Myc) is a short-lived (t1/2 = 20–30 min) nuclear onco-
protein and is a member of the basic-helix–loop-helix leucine zip-
per (bHLH–LZ) protein family of transcription factors that includes
its obligate binding partner, Max, and its antagonist proteins of the
Mad family.1 Myc is responsible for the transcription of multiple
target genes involved in cell cycle progression, cell proliferation,
differentiation, metabolism and survival.1–4 Though Myc is essen-
tial for normal cell function, its dysregulation is associated with a
wide variety of human cancers, including lung, prostate, pancre-
atic, breast, gynecological and colon cancers, B-cell lymphomas
and leukaemias.5,6 In 2012, it is estimated there will be 577,190
cancer deaths in the United States alone,7 perhaps as many as
one-seventh of which will be associated with tumours exhibiting
changes in the c-myc proto-oncogene or its expression.1 Inactiva-
tion of Myc results in cell-cycle arrest, apoptosis and tumour
regression.8–10 Thus, the inhibition of Myc function is an appealing
tactic to expand the arsenal of anticancer therapeutics in a highly
targeted manner. Currently, there is no such ‘Myc drug’ in the
clinic, and the most potent Myc inhibitors to date exhibit only
low micromolar IC50 values in vitro and in cells.5 Given the
significant role of Myc in the development and progression of a
wide variety of cancers, there is an urgent need for more potent
and diverse Myc inhibitors.
In its monomeric form, the bHLH–LZ domain of Myc (and Max)
is intrinsically disordered, presenting no obvious binding sites for
the development of inhibitors.11 Significantly, Myc becomes trans-
criptionally functional only upon its heterodimerization with Max,
an event in which the two proteins act as each other’s chaperone to
generate a parallel, left-handed four-helix bundle structure that
recognizes the obligate E-box DNA sequence CAC/TGTG. The
Myc–Max heterodimer thus serves as an excellent example of
the coupled folding and binding of two intrinsically disordered
proteins. Notably, all known biological activities of Myc require
this interaction.12–14 The HLH and LZ domains of Myc and Max
form the dimerization interface , whilst the basic regions (b) recog-
nize the DNA. Unlike Myc, Max is constitutively expressed, is stable
(t1/2 > 24 h) and can homodimerize. Max–Max homodimers bind
the same DNA sequence as Myc–Max heterodimers but rather
inhibit transcription, since Max lacks a transcriptional activation
domain. Small-molecules that interfere with the Myc–Max
protein–protein dimerization process would be expected to inhibit
the oncogenic activity of Myc. Indeed, several groups have identi-
fied inhibitors of Myc–Max dimerization that also inhibit the pro-
liferation of Myc-overexpressing cell lines.5,6 Owing to a lack of
recognizable binding sites, both in the Myc monomer and in the
Myc–Max heterodimer, which results in an inability to predict
the structures of potential Myc–Max disruptors, the majority of
inhibitors reported to date have been identified by screening large
libraries of compounds, albeit with minimal success rates.15–21
Moreover, most of these inhibitors have been hampered by
⇑
Corresponding author. Tel.: +1 410 706 6361; fax: +1 410 706 5017.
E-mail address: sfletche@rx.umaryland.edu (S. Fletcher).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.013

J. L. Yap et al. / Bioorg. Med. Chem. Lett. 23 (2013) 370–374
371
low-affinity binding and/or poor in vivo activity against Myc-
dependent tumors.6,22,23
Of particular interest to us are two such-identified Myc–Max
dimerization inhibitors: the rhodanine derivative (Z)-5-(4-
ethylbenzylidene)-2-thioxothiazolidin-4-one (10058-F4) and the
benzofurazan N-([1,10-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxa-
diazol-4-amine (10074-G5, 1).16 Upon further characterization, it
was recently revealed that the mode of Myc–Max dimerization
inhibition by these two small-molecules is through the direct bind-
ing of the Myc monomer (10058-F4 binds Myc402–412, 10074-G5
binds Myc363–381), thereby limiting its ability to interact with
Max.24–26 Previously, the activity of 10058-F4 was improved
through the development of a variety of congeners.27 10074-G5
also represents a promising candidate for optimization for several
reasons. First, it is accessible in a single chemical reaction from
commercially available starting materials. Second, it comprises
three distinct moieties (Fig. 1)—a 2-aminobiphenyl, a bicyclic ben-
zofurazan and a nitro group—each of which can be studied in a
step-wise manner through employing routine chemical reactions.
Thus, towards the optimization of 10074-G5, we have conducted
a structure–activity relationship (SAR) analysis of each of these
three components in order to first determine the molecule’s phar-
macophore, and our results are disclosed in this Letter.
Figure 2. Docking between 10074-G5 and Myc363-381, as described in Ref. 24
(adapted from Ref. 24, with permission from Steven J. Metallo).
and 2. Data are reported as percentage inhibitions that were deter-
mined through comparison of Myc–Max/DNA band intensities in
the presence of the candidate inhibitor relative to the control band
intensity acquired in its absence. First, as shown by the activity for
3a, an unsubstituted aniline at the 4-position of the 7-nitrobenzof-
urazan nucleus was insufficient for activity. Furthermore, a small
hydrophobic group (3e) or hydrophilic group (3g) at the ortho po-
sition afforded compounds with minimal Myc–Max inhibitory
activities. On the other hand, bulky, hydrophobic groups at the
ortho position (phenyl (10074-G5), bromo (3f)) furnished inhibi-
tors that exhibited about 40% disruption of the Myc–Max dimer.
These groups may be directed into the hydrophobic domain com-
posed of Phe375 and Ile381. Shifting the ortho-phenyl ring to the
meta and para positions (3l and 3n, respectively) led to a reduction
in activity, underscoring the importance of hydrophobicity at the
ortho site. Consistent with this is the observation that a polar car-
boxylic acid was poorly tolerated at the ortho position (3i) (and
also at the meta position (3m)). In contrast, excellent inhibition
of Myc–Max dimers was achieved with a carboxylic acid at the
para position of the aniline (3q, hereafter ‘JY-3-094’). This trend
also suggests that the promising activity of JY-3-094 is not simply
a consequence of improved solubility by virtue of the ionizable car-
boxylic acid since the regioisomers 3i and 3m would be expected
to exhibit comparable solubilities, yet they demonstrated little to
no inhibitory activities.
It has recently been demonstrated that 10074-G5 binds the Myc
peptide Myc353–437 (which encompasses the bHLH-LZ domain) in
the region Arg363-Ile381 with a Kd value of 2.8 l
M.24,25 More
particularly, NMR NOESY analysis and docking studies suggested
that 10074-G5 binds in a cavity that is created by a kink
(Asp379–Ile381) in the N-terminus of an induced helical domain
(Leu370–Arg378), with the biphenyl moiety observed in the
hydrophobic domain formed by Phe375, Ile381 and the side chain
methylenes of Arg378, whilst the electron rich 1,2,5-oxadiazole
(furazan) ring and nitro group were found near the positively-
charged residues Arg366 and Arg367 (Fig. 2). In order to determine
the pharmacophore of 10074-G5, we first needed to synthesize
several analogues. As anticipated, the requisite chemical reactions
were straightforward and are illustrated in Scheme 1. Variation of
the biphenyl moiety was accomplished by reacting 4-chloro-7-
nitrobenzofurazan (2) with a range of anilines and aliphatic amines
(RNH2) to furnish target molecules 3a–3q in moderate to excellent
yields. Hydrogenation of the nitro group of 10074-G5 provided the
corresponding aniline 4 that was subsequently reacted with
various acylating agents to deliver compounds 5a–5e. Finally,
N-arylation of 2-aminobiphenyl with 4-fluoronitrobenzene
delivered secondary aniline 6 (Supplementary data).
Our 10074-G5 analogues were initially screened at 100 lM in
an electrophoretic mobility shift assay (EMSA) that we have previ-
ously described.26 Effective compounds disrupt the interaction of
homogeneously purified recombinant Myc bHLH–LZ domain and
Max proteins with a fluorescently-tagged (FAM) E-Box-containing
double-stranded oligonucleotide. Our results are shown in Tables 1
The isosteric replacement of the aniline NH group with oxygen
(compound 3j) had no impact on the disruption of the Myc–Max
dimer, indicating that a hydrogen bond donor is probably not
important here. Reduction of the nitro group to the corresponding
amine (4) was not tolerated, and this may be a consequence of the
loss of the interaction between the electron rich nitro group and
the positively-charged Arg366 and Arg367 residues. Acetylation
of the primary amino group of 4 to furnish acetamide 5a did not
lead to recovery of Myc inhibitory activity. However, replacement
of the acetyl group with the more-electron rich trifluoroacetyl bio-
isostere delivered inhibitor 5b that effected 25% inhibition of the
Myc–Max heterodimer. Moreoever, the coupling of 4 to diglycolic
anhydride furnished N-acyl derivative 5e with a carboxylic acid
that demonstrated even greater inhibition of the Myc–Max hetero-
dimer (33%). Taken together, these data suggest the nitro group of
10074-G5 does, indeed, make contacts with positively-charged
residues, such as Arg367. Deletion of the 1,2,5-oxadiazole ring of
the bicyclic benzofurazan system to give 6 abolished activity, indi-
cating the importance of the heterocyclic nitrogen and oxygen
atoms that may be involved in polar interactions with Arg366
Figure 1. The chemical structure of Myc inhibitor 10074-G5 and its three
components that may be readily modulated in an SAR program.

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J. L. Yap et al. / Bioorg. Med. Chem. Lett. 23 (2013) 370–374
Scheme 1. (a) RNH2, CH3CN, DIPEA, reflux 16 h, 35–99%; (b) H2, 10% Pd/C, MeOH, RT, 16 h, 76%; (c) R2COX, CH2Cl2, Et3N, 0 °C to RT (or 40 °C), 16 h, 38–95%.
Table 1
EMSA-Based SAR analysis of 2-aminobiphenyl moiety of Myc inhibitor 10074-G5
and Arg367 as originally proposed.24,25 The nitro group and ben-
zofurazan bicycle, therefore, appear critical for Myc inhibitory
activity.
On the basis of the EMSA screening data, we can, thus, define
the pharmacophore of 10074-G5 as follows. Maximal Myc inhibi-
tory activity requires the electron rich 7-nitrobenzofurazan ring,

J. L. Yap et al. / Bioorg. Med. Chem. Lett. 23 (2013) 370–374
373
Table 2
EMSA-Based SAR analysis of 7-nitrobenzofurazan moiety of Myc inhibitor 10074-G5
Figure 3. JY-3-094 exhibits a dose-dependent inhibition of the Myc–Max heterodimer, with no effect against Max–Max homodimers, as demonstrated by (A) a reduction in
the Myc–Max/DNA band intensity and (B) no change in the Max–Max/DNA band intensity in EMSA assays (for further details, see Supplementary data).
which is consistent with its predicted binding mode to the posi-
fivefold as potent as the original lead 10074-G5 (IC50 = 146
Moreover, JY-3-094 showed no activity against Max–Max homodi-
mers at 100 M (Figs. 3 and 4), demonstrating a greater than three-
lM).
tively-charged residues Arg366 and Arg367 that would be ex-
pected to contribute considerably to the binding energy.
Furthermore, the 7-nitrobenzofurazan should be substituted with
a secondary aniline (or phenol) at the 4-position. More particularly,
this aniline requires a bulky, hydrophobic substituent (e.g., phenyl)
at the ortho position, which may engage in van der Waals interac-
tions with Phe375 and Ile381, or a carboxylic acid at the para posi-
tion, which may be involved in a salt bridge with Arg378 that is
located at the N-terminus of the induced helix. We believe this
information may prove useful in the development of future cong-
eners of 10074-G5.
l
fold selectivity for Myc–Max heterodimers, and suggesting that the
mode of inhibition of JY-3-094 is through binding Myc rather than
Max or DNA. Indeed, a mixture of JY-3-094 and the ds oligo used in
the EMSA assay were incubated together, then subjected to PAGE;
no binding of the small-molecule to the DNA was observed. We
were next keen to investigate the activity of JY-3-094 in Myc-over-
expressing cell lines. Daudi Burkitt lymphoma cells and human
promyelocytic HL60 leukaemia cells both express high levels of
Myc.16 The lead compound 10074-G5 performs well in these cell
Upon further characterization, JY-3-094 was shown to exhibit a
dose–response inhibitory effect against Myc–Max dimerization
(Fig. 3), with an IC50 value of 33 lM (Fig. 4), which is around
lines, with IC50 values of about 30
10
M in Daudi cells.16 Unfortunately, JY-3-094 demonstrated lim-
ited cytotoxicities toward these cell lines, with IC50s of 184
lM in HL60 cells and about
l
lM

374
J. L. Yap et al. / Bioorg. Med. Chem. Lett. 23 (2013) 370–374
Chemical Society Pre-Doctoral Medicinal Chemistry Fellowship
(JLY) and NIH grant R01 CA140624 (EVP).
Supplementary data
Supplementary data associated (Experimental procedures and
characterization data (1H NMR, 13C NMR and LRMS) of compounds
10074-G5, 3a–3q, 4, 5a–5e and 6) with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
013.
References and notes
Figure 4. JY-3-094 is selective for Myc–Max heterodimers over Max–Max homo-
dimers, as determined by EMSA assays.
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Acknowledgments
Financial support for this work is gratefully acknowledged from
the University of Maryland School of Pharmacy (SF), an American